O'Higgins dropped to third in the table after their shock loss at Huachipato, who claimed a second consecutive victory to rise to 13th.
Huachipato had lost three in a row before last week's 1-0 win over Nublense and again tasted victory on Saturday thanks to Francisco Arrue's 25th-minute penalty.
Despite taking 11 shots to Huachipato's seven, O'Higgins could not find an equaliser and the hosts held on for the three points.
That left O'Higgins seven points adrift of Colo Colo, who had to survive a nervous few final minutes against Cobresal.
Colo Colo took the lead on Friday in the 10th minute when Esteban Paredes struck his seventh goal in his past six matches, running onto a long ball and guiding a left-footed shot into the net.
Cobresal equalised from the spot in the 24th minute after the referee spotted a handball in the box with Ever Cantero converting the penalty but, two minutes into the second half, Colo Colo took the lead again in controversial circumstances.
Juan Delgado's header from a corner was saved by Cobresal's Leonel Cuerdo but the hosts' midfielder followed up and stabbed out of the goalkeeper's hands and into the net.
Despite the visitors' protests, the goal stood and Colo Colo extended their lead with 26 minutes left when Felipe Flores won the ball from Cobresal defender Wladimir Herrera converted past Cuerdo.
Although Lino Maldonado struck in the 90th minute for Cobresal, the visitors were unable to find an equaliser and Colo Colo held on for their sixth win in a row.
Colo Colo lead the Primera Division with 25 points, six ahead of Universidad Catolica, who defeated Deportes Iquique 1-0.
Palestino moved within two points of O'Higgins in fourth spot with a 1-0 victory at Cobreloa on Sunday, while in the other late matches of the round Nublense defeated Rangers 1-0 and Everton trumped Santiago Wanderers 2-1.
In other results, Universidad de Chile gave up a 2-1 lead and then fought back from 3-2 down to draw 3-3 with Union Espanola, Union La Calera triumphed 3-2 at Antofagasta and Audax Italiano played out a 2-2 draw with Universidad Concepcion.
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